Alzheimer's Disease (AD) is the most common neurodegenerative disorder of aging, and is characterized by progressive dementia and personality dysfunction. The abnormal accumulation of amyloid plaques in the vicinity of degenerating neurons and reactive astrocytes is a pathological characteristic of AD.
Several lines of studies suggest that postmenopausal women with lower levels of endogenous estrogen may be predisposed to the development of AD. Studies in experimental animal models provide a convincing rational for the role of estrogen replacement therapy and prevention of dementia. See, for example, Birge, J. Am. Geriatric. Soc. 44, 865, (1996). These studies suggest that estrogen deficiency in postmenopausal women apparently increases their susceptibility to the neurodegenerative changes of aging and AD, and that this risk can be decreased by estrogenic replacement therapy. Peganini et al. Am. J. Epidemiol, 140, 256 (1994). Estrogen treatment of cell culture reportedly promotes non-amyloidogenic APP processing and soluble APP (APPs) secretion. Jaffe et al., J. Biol. Chem. 269, 13065 (1994).
APP processing is regulated by neurotransmitters and synaptic activity. Amyloid plaques in AD accumulate near dystrophic neurons and reactive astrocytes. Cordell, Annu. Rev. Pharmacol. Toxicol. 34, 69 (1994); Selkoe, Annu. Rev. Neurosci. 17, 489 (1994). The activation of neurotransmitter receptors, which are coupled to phosphotidylinositol (PI) hydrolysis or to protein kinase C (PKC) activation, can promote APP metabolism and decrease amyloid formation. Nitsch, et al., Science 258, 304 (1992); Wolf et al., J. Biol. Chem. 270, 4916 (1995); Buxbaum et al., Proc. Natl. Acad. Sci. U.S.A. 91, 4489 (1994); Lee, et al., Ibid., 92, 8083 (1995); Ulus and Wurtman, J. Pharm. Exp. Ther., 281,149 (1997); Lee et al., Proc. Natl. Acad. Sci. U.S.A. 92, 8083 (1995). Activation of neurotransmitters coupled to cAMP production suppresses both constitutive and PKC/PI-stimulated APPs secretion in astroglioma cells and in primary astrocytes. Eftimiopoulos et al., J. Neurochem., 67, 872 (1996); Lee et al., J. Neurochem., 68,1830 (1997). The drastic alterations in neurotransmitter levels and second messenger signaling created by neurodegeneration and synapse loss in AD may disrupt APP processing in ways that promote the accumulation of amyloidogenic or neurotoxic APP fragments.
Additionally, the loss of various neurotransmitters in AD may increase cellular levels of APP holoprotein containing amyloidogenic or neurotoxic peptides due to a decrease in proper APP metabolism. Yankner et al., Science, 245, 417 (1989); M. R. Kozlowski, A. Spanoyannnis, S. P. Manly, S. A. Fidel, R. L. Neve, J. Neurosci, 12, 1679 (1992).
Increased APP production in Down's syndrome is associated with a high incidence of AD at an early age due to the extra copy of the APP gene. Over expression of APP in cell cultures and in transgenic mice is also associated with neurodegeneration and age-related cognitive deficits, suggesting that over expression of APP could contribute to the neuropathology of AD. Maruyama, et al., Nature, 347, 566 (1990); Hsiao et al., Neuron 15, 1203-1218 (1995); Moran, et al., Proc. Natl. Acad Sci. U.S.A. 92, 5341 (1995).
Several APP isoforms, ranging in size from 695-770 amino acids, are derived by differential splicing of a primary transcript of the three major APP isoforms, APP-695 is predominantly expressed in neurons; APP-751 and APP-770, which harbor an additional Kunitz-type protease inhibitor (KPI) insert at the N-terminus, are predominantly expressed in astrocytes and appear to be increased in AD brain. Golde, S. et al., Neuron 4, 253 (1990); Neve, et al.,Ibid., 1, 669 (1990); Anderson et al., EMBO J. 8, 3627 (1989); Nordstedt et al., Proc. Natl. Acad. Sci. U.S.A. 88, 8910 (1991). The decreased amounts of APP-695 in postmortem AD brains may be due to neuronal loss. The increase in KPI-containing APP isoforms in AD and in regions surrounding senile plaques raises the possibility that APP overexpression in astrocytes contributes to AD neuropathology.
Several lines of studies have indicated an apparently low incidence of Alzheimer's Disease in rheumatoid arthritis patients. These studies propose the possibility that anti-inflammatory therapy with NSAIDs (non-steroidal anti-inflammatory drugs) confers some protection against AD. See, e.g., McGeer, et al., Lancet, 335, 1037 (1990); Andersen, et al., Neurology, 45, 1441 (1995); Rich, et al. Neurology, 45, 51 (1995); Lindsay, et al., Neurology, 44, 2073 (1994); Rogers, et al., Neurology, 43, 1609 (1993). These studies suggest that the administration of a non-steroidal anti-inflammatory drug may reduce the onset of dementia in Alzheimer's patients. However, none of these studies discloses or suggests that the administration of NSDAs prevents the overproduction of APP.
Persistent and rapid elevations in APP immunoreactivity have been observed in GFAP-positive astrocytes following brain injury. Siman et al., J. Neurosci., 3, 275 (1989); Banati and Kreutzberg, J. Cereb. Blood Flow Metab., 12, 257 (1995). In the AD brain, the loss of synapses is associated with an increase in the number of GFAP-positive astrocytes (Brun et al., Neurodegeneration, 4, 171 (1995), and increases in KPI containing APP mRNA in the frontal cortex have also been attributed to the astrocytic response during neuronal damage (Golde et al., Neuron, 4, 253 (1990). It seems that the loss of synapses and neurons in AD might initiate a pathological cascade that includes APP overexpression by reactive astrocytes.
In U.S. Pat. No. 5,385,915, Buxbaum et al. describe methods and compositions for affecting APP processing by the administration of agents that regulate protein phosphorylation, namely agents that regulate kinases or phosphatases. The modulation of APP processing leads, in turn, to the regulation of the production of .beta./A.sub.4 peptide, a peptide that accumulates in amyloidogenic plaques. See, e.g., col. 6, lines 8-10. Hence, Buxbaum et al. teach that one's objective should focus on the search for agents that alter the metabolism of APP.
However, Buxbaum et al. make no mention, teaching, or suggestion that the step preceding the processing of APP, that is, the expression, production, or formation of APP, itself, can be at all affected by select groups of substances. Indeed, as Buxbaum et al. state (at col. 21, lines 7-9), "the effects observed are attributable to changes in APP metabolism rather than APP transcription (emphasis added)." Consistent with this notion, the claims of Buxbaum et al. are drawn to a method of regulating phosphorylation of proteins that control the processing of APP.
Similarly, in U.S. Pat. No. 5,242,932, Gandy et al. disclose and claim a method of modulating or affecting the intracellular trafficking and processing of APP in the mammalian cell.
For additional background information on the processing of APP, release of APP derivatives, or the processing, degradation and secretion of .beta./A.sub.4 APP, the interested reader is referred to the following publications: Nitsch, et al. Science, 258, 304 (1992); Lee, et al., Proc. Nat'l. Acad. Sci. USA, 92, 8083 (1995); Caporaso, et al. Proc. Nat'l. Acad. Sci. U.S.A, 89, 3055 (1992); Caporaso, et al. Proc. Nat'l. Acad. Sci. U.S.A, 89, 225 (1992); and Buxbaum, et al. Proc. Nat'l. Acad. Sci. U.S.A, 89, 10075 (1992).
In contrast to the above studies, the present invention, as disclosed herein, concerns the expression, formation, or synthesis of APP. The present inventors have previously discovered that cAMP elevations caused by activation of neurotransmitter receptors increased APP mRNA and holoprotein production in astrocytes. Lee et al.,et al. Proc. Nat'l. Acad. Sci. U.S.A, 94, 5422 (1997). The present inventors have also shown that activation of prostaglandin E.sub.2 (PG E.sub.2) receptors coupled to increased cAMP formation also stimulates the synthesis of APP mRNA and holoprotein and that this effect can be mediated by cAMP-dependent protein kinases, and can be inhibited by various substances, including immunosuppressants and ion-channel modulators. Lee et al., J. Neurochem. (supp) 69, S103B (1997). As described herein, the present inventors have now shown that APP expression can also be regulated by lipophilic hormones that interact with cytosolic or nuclear receptors.
Estrogen receptors within the brain have a regional distribution strikingly similar to that characteristics of AD-type brain pathology, Thomlinson, B. E.(1992): In Greenfield's neuropathology (Adams, J. H. and Duchen L. W. eds)pp 1284-1410 Oxford University Press. In addition recent findings suggest that estrogen replacement therapy my be protective against AD, Henderson, et al., Arch neurol (1994). In order to test the hypothesis that gonadal esteroids might play a role in regulating APP metabolism, Jaffe, et al., J. Biol. chem.269, 13065 (1994) investigated the possible effect of estrogen on the metabolism of Alzheimer's amyloid precursor protein. Using a cell line that contains high level of estrogen receptors, these authors found that the treatment with 17.beta.-estradiol is associated with the accumulation of APPs in the medium, indicative of non-amyloidogenic processing. However, these authors found no changes in the levels of intracellular immature or mature APP holoproteins, suggesting that estrogen may increase the secretory metabolism of APP.
Additionally, Hsiao et al., Neuron, 15, 1203 (1995), disclose that age-related central nervous system (CNS) disorder and early death in mice were due to over expression of APP, rather than amyloid, with increasing level of intracellular APP, indicating that some deleterious processes related to APP over expression are dissociated from formation of arnyloid.
Previously, it has been shown that neurotrophic effect of estrogen may be through uncoupling signal transduction mechanism of certain receptors linked to G-proteins. Lagrange, et al., Soc. Neursci. Abstr. 22, 378, (1996). Additionally, the present inventors have discovered that G-protein coupled receptors which stimulate cAMP formation or PI hydrolysis can accelerate the synthesis and metabolism of APP respectively.
This invention, as disclosed herein, demonstrates for the first time that lipophilic compounds such as estrogen affects the synthesis of the APP holoprotein. The present invention also contemplates and provides an assay for identifying or screening potential drugs that can inhibit the excessive production of neurotoxic APP. For example, such an assay may utilize a tissue or cell culture comprising brain cells, as described herein.
These and other objects of the invention will be evident to those of ordinary skill from a consideration of the discussions and descriptions provided in this specification, including the detailed description of the preferred embodiments.